Pathogen induced infections in humans, animals, and tissue culture have generally been treated with anti-infective agents such as antibiotic, antimicrobial, antiviral or antifungal agents, depending on the type of pathogen involved. However, these agents have led to detrimental selection of pathogens that have become resistant to these administered agents. Today, infectious disease specialists now speak openly of a coming post-antibiotic era as previously well-controlled organisms have now become resistant to commonly available antibiotics. For example, Staphylococcus aureus is now widely resistant to methicillin. Methicillin-resistant S. aureus has been treated with vancomycin, however, the transfer of glycopeptide resistance from Enterococci into S. aureus species occurs readily in vivo, pointing to a short therapeutic life span of this drug. Although inappropriate use contributes to the rise in antibiotic resistance, it is also clear that resistant bacteria are selected when antibiotics are used prudently. Beyond this trend, opportunistic infections of bacterial, fungal and viral etiology have emerged in immunocompromised patients, especially in those infected by HIV.
The rise in antibiotic resistance can be slowed by changing patterns of antibiotic use or by using combination drug therapy. But it is also necessary to develop new anti-infective agents, as, for example, several microbes have been found to express multiple antibiotic resistance phenotypes which can be resistant to multiple treatment regimens and to antibiotics which are unrelated in chemical structure. The appearance of such bacteria and infections by such bacteria greatly increase the difficulty of identifying effective antibiotics and treating infections in humans or other animals, and in cell culture.
Although it is possible to use rational drug design to identify new anti-infective agents, the more common and fruitful strategy has been to screen large compound libraries for their activity against molecular targets. Recent efforts in combinatorial chemistry, combinatorial genetics and natural product chemistry address the need for larger libraries and for more efficient identification of active compounds. The expanding diversity of compounds for screening has not been matched by an equal expansion of targets. Current efforts in genomic sequencing are among the strategies being employed to identify new targets for screening.
Genomic sequence information is expected to lead to the identification of new targets for rational design of potential therapeutics. Thus, for example, genomic sequencing of pathogens' genomes has revealed widespread occurrence of “pathogenicity islands,” that is, clusters of genes, likely spread through horizontal transmission, whose (often conditional) expression gives pathogenic ability to their bacterial host. The ability to pharmacologically intervene in this state would be extremely valuable. However, target identification can be the rate-limiting step in high-throughput screening, even with the extensive genomic information determined within the past five years. Although it is possible to identify targets based on well-characterized model biochemical and genetic systems, not all gene functions are known, even those of well-studied organisms. For example, no biochemical function has been assigned for 30% of open reading frames in the genome sequence of Escherichia coli (E. coli), the paradigm organism of molecular biology. One possible strategy for drug screening would start by fully determining the roles of these functions, however, the preliminary characterization required is labor-intensive and time-consuming.
A further paradigm for genome-driven pharmaceutical discovery is identifying inhibitors of lipid A synthesis. Extensive genetic studies identified lipid A synthesis as essential for growth, compounds were identified that inhibited galactose incorporation into bacteria, the target was identified as a unique deacetylase and the potency of the original compound was improved through a synthetic chemistry program.
Genomic information has the ability to streamline the search the extensive genetic studies that precede the screening and medicinal chemistry portions of a drug-discovery program. A promising approach is to identify genes on the basis of their phylogenetic conservation only. Phylogenetically conserved proteins, especially those that are conserved across all bacteria, for example, are likely more essential for microbial survival than genes that are either (1) unique to a few bacteria, or (2) common across all taxa. Neither of these assumptions has been rigorously tested, although Arigoni et al. (Arigoni F., et al., 1998. Nature Biotechnology 16:851-856) systematically tried to knock out 26 open reading frames of unknown function that are common to E. coli and M genitalium. Only six open reading frames were essential for growth in culture, as judged by the failure to recover the knockouts.
More recently, the open reading frames predicted from all known genomic sequences have been organized into Clusters of Orthologous Groups (COGs). The Clusters of Orthologous Group database is a tool in identifying molecular targets for screening. Orthologous protein sequences are derived from a common ancestor and appear in different species, as opposed to paralogues which arise from gene duplication within a species. The Clusters of Orthologous Group approach starts with BLAST comparison of all the open reading frames against all other open reading frames in the database, followed by classification of all the cross-species comparisons to identify those that appear in phylogenetically distinct species. Clusters of Orthologous Groups, therefore, represent the set of all phylogenetically conserved functions. The proteins within a single Clusters of Orthologous Group have related functions and very similar secondary structures.
One application of the Clusters of Orthologous Group database to yield new biochemical data has been published, see, Riley M. and Labedan B., 1997, Journal of Molecular Biology 268:857-868. The Clusters of Orthologous Group database was used to identify protein families that lack structurally characterized members. The structure of one of the proteins from a previously uncharacterized family was shown to have an alpha-beta plait topology, even though it does not exhibit any obvious sequence similarity to known members of this structural class.
Another such protein within the Cluster of Orthologous Group is ribonuclease P (COG0594). Prokaryotic ribonuclease P is a membrane-bound ribonucleoprotein ribozyme. Several antibiotics active against protein synthesis target ribonuclease P, but these compounds primarily target the ribosome and not ribonuclease P. For example, puromycin, the toxic-antibiotic that causes premature peptide chain termination inhibits the catalytic reaction of ribonuclease P RNA, albeit at a much higher concentration than required to inhibit protein synthesis (Vioque A., 1989. FEBS Letters 246:137-139). Neomycin B inhibits the action of E. coli ribonuclease P RNA at micromolar concentrations whether of not it is complexed with the protein subunit (Mikkelsen N. E., et al., 1990. PNAS 96:61 55-6160).
A limitation of these and similar informatics-based approaches is that the Orthologous Groups are not necessarily required for bacterial viability under laboratory conditions. For example, only eight of 26 genes identified by such an approach proved to be essential when the chromosome was disrupted at these loci. Furthermore, it is likely that many essential functions were missed in this analysis. Paralogous functions (those arising from gene duplications, and therefore represented more than once in a genome) are systematically excluded by such an approach. For example, the E. coli genome codes for paralogues of gyrA and gyrB. Search criteria that did not allow paralogues would exclude these as targets for drug discovery, despite the fact that they are targets for quinolines and novobiocin, respectively.
Resistance to an inhibitor can come about by amplification of the target which can titrate the effects of an inhibitor. While the original observation of the phenomenon depended on chromosomal mutation to increase the amount of the gene product, a multicopy situation is more easily realized by cloning the relevant function in a high-copy vector. This strategy has been used in analyses of several bacterial genes and operons, however, it appears not to have been widely used, if at all, for identifying targets for antimicrobials.
Published information regarding the use of bacterial genomics in the search for new anti-infectives has been confined mostly to cases where genomic information has been applied in a “top-down” fashion. Thus, individual genes have been targeted based on analysis of common distribution among genomes whose sequences are known.
The phenomenon of multicopy suppression identifies the targets for gene functions in the absence of detailed physiological, metabolic or genetic information. Multicopy suppression is widely used to genetically identify interacting macromolecules. Interacting molecules have been identified in yeast and bacteria, and multicopy suppression useful in identifying weakly interacting or poorly expressed molecules, see, for example, Hara H. et al., 1996. Microbial Drug Resistance 2:63-72. Also, see, Danese P N. et al., 1995. Journal of Bacteriology 177 (17):4969-73. For another example, see Ueguchi C. and Ito K 1992. Journal of Bacteriology 1 74:1454-61. Also see, Berg C M. et al., 1988. Gene 65(2):195-202).
The RNA processing enzyme ribonuclease P is present in all cells and organelles that carry out tRNA biosynthesis. It cleaves the 5′ end of precursor tRNA to generate the 5′ end of mature tRNA in both prokaryotic and eukaryotic. In Escherichia coli, RNaseP was shown to participate in processing of rRNA precursors as well. RNaseP is unique among all the tRNA processing enzymes in having an RNA motif that is required for its function.
In all bacteria investigated thus far, it has been found that the enzyme is composed of two subunits, a small (about 14 kDa) protein and a large (about 130 kDa) RNA. The RNA subunit, RNaseP RNA, exhibits considerable variability in size among different species ranging from 140-490 nucleotides in length.
Previous work with E. coli, Bacillus subtilis and other bacteria showed that the RNA subunits from gram negative and positive bacteria can catalyze the cleavage of appropriate substrates in vitro, in buffers containing >20 mM Mg+2. In other words, all of the specific catalytic residues required for the function of RNaseP reside in these RNA subunits.
Both monovalent (K+ or NH4+) and divalent (Mg2+ and Ca2+) cations are of critical importance in the RNaseP reaction. Higher ionic strength than those which are optimal for the native ribozyme can suppress the affect of many mutations in the RNA only reaction, apparently by stabilizing the structure of RNA.
In addition to its structural role, Mg2+ is proposed to promote catalysis by activating a water molecule to hydrolyze the susceptible phosphodiester bond in the substrate RNA and also coordinating on phosphate oxygens.
The stoichiometry of subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme is about 4×10−10 M. However the dissociation constant of 2×10−8 to 6×10−8 M was observed when C5 protein interacts with various RNA molecules in a nonmanner.
Mammals express multiple isoforms of RNaseP RNA (Li and Williams, 1995). Three novel genes encoding small RNAs homologous to human and mouse RNaseP RNA have been isolated from a mouse genomic library. In addition, similar short homologues of RNaseP are expressed in rate, rabbit and human cells.
The protein cofactor of RNaseP from E. coli (C5 protein) is a molecule of 119 amino acids, with a molecular mass of 13,700 daltons. Although this protein is identical in size to the C5 protein from the B. subtilis RNaseP (P-protein), there is only 25% homology between the primary sequence of the two proteins. In addition, both C5 and P-Proteins can be mixed with the heterologous P and M1RNAs to form functional hybrid holoenzymes. In addition, diverse bacterial RNA's assemble with the protein subunit of E. coli and diverse protein subunits assemble with the RNA compound of E. coli. Therefore, RNaseP holoenzyme share common features in their assembly pathway.
The C5 protein moiety functions as an electrostatic shield that allows two negatively charged RNA molecules (the catalytic subunit and the substrate) to interact. Furthermore, the presence of the protein in vitro increases the rate of cleavage reaction by RNaseP holoenzyme 20 fold.
It has been shown that at high ionic strength the cleavage reaction is protein independent, but the enzymatic turnover is slow. One of the important affects of high salt concentration is to facilitate substrate binding in the absence of protein probably by decreasing the repulsion between polyanionic enzyme and substrate RNAs. In addition to its function as a counter ion, the protein subunit facilitates substrate binding without interfering with rapid product release. In addition the protein subunit can stabilize the structure of RNA and thereby suppress the affect of many mutations that affect the RNA only reaction.
To understand the function of RNaseP it is important to elucidate how this enzyme recognizes its substrate. It is known that the tertiary folding of tRNA moiety of tRNA precursor molecule plays an important role in the enzyme substrate recognition.
The holoenzyme (RNaseP) binds to the helix formed by coaxial stacking of the common arm and acceptor stem of the tRNA, which is adjacent to the side of cleavage. In addition, the RNaseP holoenzyme recognizes the mature domain of precursor tRNA.
Work accomplished by Talbot and Altman (1994) revealed that regions of M1 RNA that interact with C5 proteins are clustered into three main areas that are localized between nucleotides 41-99, 168-198 and 266-287. Some point mutations that significantly affect the activity of M1RNA are located in these regions. Furthermore, nucleotides 254-259 and 291-195 form a binding site for a magnesium ion. The Mg2+ ion bound in this vicinity participates in the chemical step of the cleavage reaction.
Many mutants defective in RNaseP have been isolated. These mutants seem to fall into two groups. One of these two groups is exemplified by the rnpA 49 mutation. The rnpA 49 mutation in the rnpA gene, the gene coding for C5 protein in E. coli results in an arginine to histidine alteration at position 46 in the C5 protein.
Apirion (1979) found that rnpA 49 is a recessive point mutation. Apirion and Watson (1979) analyzed a second class of mutation (rnpB, gene coding for M1RNA) which affects the RNaseP function. This temperature sensitive mutation carried by the rnpB strain is located between min 64 and min 81 of the E. coli chromosome. It has been shown that the rnpA and rnpB mutation occurs in two different genes (Apirion and Watson, 1979).
Cells bearing the rnpA 49 mutation are not able to grow at non-permissive temperature (42° C.). The tRNA precursor is accumulated when these cells shifted from permissive temperature to non-permissive perature (Schedl, et al., 1975). In addition, nonRNa-functional seP can be characterized by the appearance of a 19S RNA molecule, which contains 16S rRNA and spacer tRNA, and of a number of tRNA precursor molecules, and by the disappearance of the 4.5 SRNA molecule. Work from this laboratory showed that the over expression of the rnpB gene (gene coding the RNA subunit) from a high copy vector can complement or suppress the temperature sensitive phenotype of E. coli strain bearing rnpA 49.
Baer, et al. (1989) found that the temperature sensitive phenotype of cell bearing the A49 mutation can be suppressed by increasing in the efficiency of assembly of the holoenzyme in vivo. Moreover, in the presence of excess M1RNA the cleavage activity of reconstituted holoenzyme from wild type M1RNA and C5A49 increases in vitro as well as in vivo (Baer, et al., 1989). (Baer et al., 1989) also found that the Arg-46 to His-46 mutation in the C5A49 protein affects the ability of the protein to participate with M1RNA in the normal assembly process of RNaseP.
In addition, a study by Morse & Schmidt (1993) showed that the catalytic activity of different RNA's mutant (M1RNA) are similar to wild type RNA. However, strains of E. coli bearing these mutations cannot support cell growth in vivo which indicates that some steps other than catalysis must be responsible for the loss of biological function in vivo. Several experiments have suggested that assembly of RNaseP is deficient in these mutants. Thus the assembly of the RNA moiety and C5 protein is required for cell growth.
Therefore, it would be highly advantageous to identify anti-infective agents through high-throughput screening and then to use the sequences identified through a multicopy suppression assay to define targets and, potentially, modes of resistance and compounds directed to these targets. Each such identified target can then be used in an iterative fashion to classify similar agents at an early stage of development, direct further searches for lead compounds that are focused on one or more defined targets, and identify agents that are likely to be compromised by known means of resistance, for example, drug efflux pumps. It would also be highly advantageous to have an assay that allows screens that can be directed to a novel target, that can be used to identify inhibitors of pathogenic components, that can be used in cases were cell viability depends on assembly, that can be extended to other targets, such as sensory, secretory, and regulatory bacterial macromolecular assemblies, and/or that can be used as a high throughput screening assay for inhibitors of macromolecular assembly.